Fluorescent molecules are attractive as reporter molecules in many assay systems because of their high sensitivity and ease of quantification. Recently, fluorescent proteins have been the focus of much attention because they can be produced in vivo by biological systems, and can be used to trace intracellular events without the need to be introduced into the cell through microinjection or permeabilization. The green fluorescent protein of Aequorea victoria is particularly interesting as a fluorescent indicator protein. A cDNA for the protein has been cloned. (D.C. Prasher et al., "Primary structure of the Aequorea Victoria green-fluorescent protein," Gene (1992) 111:229-33.) Not only can the primary amino acid sequence of the protein be expressed from the cDNA, but the expressed protein can fluoresce. This indicates that the protein can undergo the cyclization and oxidation believed to be necessary for fluorescence.
Aequorea green fluorescent protein ("GFP") is a stable., proteolysis-resistant single chain of 238 residues and has two absorption maxima at around 395 and 475 nm. The relative amplitudes of these two peaks is sensitive to environmental factors (W. W. Ward. Bioluminescence and Chemiluminescence (M. A. DeLuca and W. D. McElroy, eds) Academic Press pp. 235-242 (1981); W. W. Ward & S. H. Bokman Biochemistry 21:4535-4540 (1982); W. W. Ward et al. Photochem. Photobiol. 35:803-808 (1982)) and illumination history (A. B. Cubitt et al. Trends Biochem. Sci. 20:448-455 (1995)), presumably reflecting two or more ground states. Excitation at the primary absorption peak of 395 nm yields an emission maximum at 508 nm with a quantum yield of 0.72-0.85 (O. Shimomura and F. H. Johnson J. Cell. Comp. Physiol. 59:223 (1962); J. G. Morin and J. W. Hastings, J. Cell. Physiol. 77:313 (1971); H. Morise et al. Biochemistry 13:2656 (1974); W. W. Ward Photochem. Photobiol. Reviews (Smith, K. C. ed.) 4:1 (1979); A. B. Cubitt et al. Trends Biochem. Sci. 20:448-455 (1995); D. C. Prasher Trends Genet. 11:320-323 (1995); M. Chalfie Photochem. Photobiol. 62:651-656 (1995); W. W. Ward. Bioluminescence and Chemiluminescence (M. A. DeLuca and W. D. McElroy, eds) Academic Press pp. 235-242 (1981); W. W. Ward & S. H. Bokman Biochemistry 21:4535-4540 (1982); W. W. Ward et al. Photochem. Photobiol. 35:803-808 (1982)). The fluorophore results from the autocatalytic cyclization of the polypeptide backbone between residues Ser.sup.65 and Gly.sup.67 and oxidation of the .alpha.--.beta. bond of Tyr.sup.66 (A. B. Cubitt et al. Trends Biochem. Sci. 20:448-455 (1995); C. W. Cody et al. Biochemistry 32:1212-1218 (1993); R. Heim et al. Proc. Natl. Acad. Sci. USA 91:12501-12504 (1994)). Mutation of Ser.sup.65 to Thr (S65T) simplifies the excitation spectrum to a single peak at 488 nm of enhanced amplitude (R. Heim et al. Nature 373:664-665 (1995)), which no longer gives signs of conformational isomers (A. B. Cubitt et al. Trends Biochem. Sci. 20:448-455 (1995)).
Fluorescent proteins have been used as markers of gene expression, tracers of cell lineage and as fusion tags to monitor protein localization within living cells. (M. Chalfie et al., "Green fluorescent protein as a marker for gene expression," Science 263:802-805; A. B. Cubitt et al., "Understanding, improving and using green fluorescent proteins," TIBS 20, November 1995, pp. 448-455. U.S. Pat. No. 5,491,084, M. Chalfie and D. Prasher. Furthermore, engineered versions of Aequorea green fluorescent protein have been identified that exhibit altered fluorescence characteristics, including altered excitation and emission maxima, as well as excitation and emission spectra of different shapes. (R. Heim et al., "Wavelength mutations and posttranslational autoxidation of green fluorescent protein," Proc. Natl. Acad. Sci. USA, (1994) 91:12501-04; R. Heim et al., "Improved green fluorescence," Nature (1995) 373:663-665.) These properties add variety and utility to the arsenal of biologically based fluorescent indicators.
There is a need for engineered fluorescent proteins with varied fluorescent properties.